An efficient method for the construction of artificial, concatemeric DNA, RNA and proteins with genetically programmed functions, using a novel, vector-enzymatic DNA fragment amplification-expression technology
Piotr M. Skowron,
Natalia Krawczun,
Joanna Żebrowska,
Daria Krefft,
Olga Żołnierkiewicz,
Marta Bielawa,
Joanna Jeżewska-Frąckowiak,
Łukasz Janus,
Małgorzata Witkowska,
Małgorzata Palczewska,
Agnieszka Zylicz-Stachula
Affiliations
Piotr M. Skowron
Department of Molecular Biotechnology, Faculty of Chemistry, University of Gdansk, Gdansk 80-308, Poland; BioVentures Institute Ltd., Poznan 60-141, Poland; Corresponding author at: Department of Molecular Biotechnology, Faculty of Chemistry, University of Gdansk, Gdansk 80-308, Poland.
Natalia Krawczun
Department of Molecular Biotechnology, Faculty of Chemistry, University of Gdansk, Gdansk 80-308, Poland; BioVentures Institute Ltd., Poznan 60-141, Poland
Joanna Żebrowska
Department of Molecular Biotechnology, Faculty of Chemistry, University of Gdansk, Gdansk 80-308, Poland; BioVentures Institute Ltd., Poznan 60-141, Poland
Daria Krefft
Department of Molecular Biotechnology, Faculty of Chemistry, University of Gdansk, Gdansk 80-308, Poland; BioVentures Institute Ltd., Poznan 60-141, Poland
Olga Żołnierkiewicz
Department of Molecular Biotechnology, Faculty of Chemistry, University of Gdansk, Gdansk 80-308, Poland
Marta Bielawa
BioVentures Institute Ltd., Poznan 60-141, Poland
Joanna Jeżewska-Frąckowiak
Department of Molecular Biotechnology, Faculty of Chemistry, University of Gdansk, Gdansk 80-308, Poland; BioVentures Institute Ltd., Poznan 60-141, Poland
Łukasz Janus
Department of Molecular Biotechnology, Faculty of Chemistry, University of Gdansk, Gdansk 80-308, Poland; BioVentures Institute Ltd., Poznan 60-141, Poland
Małgorzata Witkowska
Department of Molecular Biotechnology, Faculty of Chemistry, University of Gdansk, Gdansk 80-308, Poland
Małgorzata Palczewska
Department of Molecular Biotechnology, Faculty of Chemistry, University of Gdansk, Gdansk 80-308, Poland
Agnieszka Zylicz-Stachula
Department of Molecular Biotechnology, Faculty of Chemistry, University of Gdansk, Gdansk 80-308, Poland; BioVentures Institute Ltd., Poznan 60-141, Poland
De novo designed bioactive molecules, such as DNA, RNA and peptides, are utilized in increasingly diverse scientific, industrial and biomedical applications. Concatemerization of designed DNA, RNA and peptides may improve their stability, bioactivity and allow for gradual release of the bioactive molecule at the intended destination. In this context, we developed a new method enabling the formation of DNA concatemers for the production of artificial, repetitive genes, encoding concatemeric RNAs and proteins of any nucleotide and amino-acid sequence. The technology recruits the Type IIS SapI restriction endonuclease (REase) for assembling DNA fragments in an ordered head-to-tail-orientation. Alternatively, other commercially available SapI isoschizomers can be used: LguI and thermostable BspQI. Four series of DNA vectors dedicated to the expression of newly formed, concatemeric open reading frames (ORFs), were designed and constructed to meet the technology needs.• Vector-enzymatic DNA fragment amplification technology.• Construction of DNA concatemers many times longer than those available with the use of current de novo gene synthesis methods.• Biosynthesis of protein tandem repeats with programmable function never seen in nature.
